Retroviruses are serious pathogens that cause immune deficiency syndromes, cancer, and neurological disease in humans. Conversely, retroviruses are highly useful for directing beneficial genes into human cells. Well characterized retroviral proteins can often be targeted by drugs or be manipulated genetically to achieve important biomedical advances. In strong contrast, retroviruses have RNA genomes and very little is known regarding RNA structures that play critical roles in retroviral infection and morbidity. One compelling example is that retroviral RNA genomes both enter and leave a targeted cell as a stable dimer. The two strands of a retroviral RNA genome are linked at their 5'ends by a precise, but poorly understood, three-dimensional structure. Understanding the mechanism of retroviral dimerization at a molecular level therefore represents opportunities both to disrupt the infectivity of pathogenic viruses and to enhance the function of therapeutic vectors. This work thus extends from basic mechanistic insights to potential clinical applications. Our team of virologists and chemical biologists will tackle the following Aims: (1) Refine a three-dimensional model for the dimerization domain of Moloney murine sarcoma virus. Analysis of this model will provide important new information regarding the RNA conformational changes that accompany dimerization and will facilitate identification of structural targets for anti-retroviral agents. (2) Determine the folding pathway for the Moloney murine leukemia virus (MuLV) RNA dimerization at nucleotide resolution in the presence and absence of the viral Gag protein. We will explore the hypothesis that dimerization progresses by highly specific sequential interactions involving distinct RNA domains and that this step-wise assembly is crucial for the ability of the virus to package its genomic RNA with such extraordinary specificity. (3) Use high-throughput RNA structure analysis technology, invented in our laboratory, to analyze (i) the structure of the dimerization domain and (ii) protein-RNA interactions inside authentic MuLV virions. This information will then be used to define, and genetically validate, the packaging signal of MuLV in vivo at single nucleotide resolution. (4) Initiate a program to determine the structure of the dimerization domain and sites of specific viral protein binding in HTLV-1. This Aim further extends our successful invention, in collaboration with retrovirologists, of direct approaches for analyzing retroviral RNA structure ex virio and in virio that circumvent the requirement for laborious and notoriously inefficient manipulation and transfection of HTLV-1. Experimental innovations developed in this Aim will be broadly useful to the virology community for analyzing any RNA structure in any RNA virus. PUBLIC HEALTH RELEVANCE: This work will advance significantly our understanding of the structure and function of the RNA genomes of retroviruses. This project makes use of multiple, innovative, advances in technology and involves a distinctive and close collaboration between chemical biologists and virologists. Over the long term, improved, molecular quality, information about retroviral genome structure has significant potential to provide a framework for designing new anti-retroviral therapeutics and for engineering better gene therapy vectors for introducing corrective genes into human cells.